Trifluorinated-1α,25S-dihydroxy vitamin D3 compounds

ABSTRACT

26, 26, 26-Trifluoro-1 alpha , 25-dihydroxy-cholecalciferol, and the 25R or 25S epimer thereof, are described, as well as processes and intermediates utilized to prepare the same, as well as pharmaceutical compositions containing the same. 26,26,26-trifluoro 1 alpha , 25-dihydroxycholecalciferol and the 25R or 25S epimer thereof are useful agents in the treatment of disease states such as osteoporosis, oseodystrophy and leukemia.

This is a division of application Ser. No. 06/821,299, filed Jan. 22,1986, now U.S. Pat. No. 4,906,785 which is a continuation-in-part ofapplication Ser. No. 06/683,442, filed Dec. 19, 1984 abandoned.

BACKGROUND OF THE INVENTION

It has been known in the art to introduce fluorines on the 26 and 27carbon atom in certain vitamin D₃ metabolites in order to enhancevitamin D like activity. Thus, for example, U.S. Pat. No. 4,358,406describes 26,26,26,27,27,27-hexafluoro-1α,25-dihydroxycholecalciferolswhich are indicated to have greater vitamin D-like activity compared tothe analogous unfluorinated compounds. The compounds of the patent areindicated to be useful in human and veterinary medicine for treatment ofcalcium and phosphorus deficiency or imbalance. These compounds are thususeful in the treatment of hypoparathyroidism, pseudohypoparathyroidism,renal osteodystrophy, osteoporosis and other bone disorders symptomaticof calcium and phosphorus imbalance. Veterinary applications include,for example, treatment of milk fever in cattle, leg weakness in turkeys,chickens and other domestic animals. Indicated therapeutic dosages forthe above indications ranged from 0.1 to micrograms/day orally orparenterally.

In U.S. Pat. No. 4,298,791 there is disclosed25-hydroxy-26,26,26,27,27,27-hexafluorocholecalciferol. This compound isindicated to have excellent vitamin D₃ -like activity as measured byability to stimulate calcium transport in the intestine and to mobilizecalcium from bone and in its antirachitic activity. Thus the compound isuseful in treatment of disease resulting from calcium metabolismdisorder.

Kobayashi et al., Tetrahedron Letters, 22 (No 43), 4309 (1981) describethe synthesis of 26,26,26-trifluoro-25-hydroxyvitamin D₃ and27-nor-26,26,26-trifluoro-25-hydroxyvitamin D₃. No biological activitywas reported although the purpose of synthesis was to compareboneresolving activity with the previously described hexafluoro analogsof vitamin D₃.

SUMMARY OF THE INVENTION

The present invention relates to novel trifluorocholecalciferolderivatives that exhibit enhanced vitamin D₃ -like activity. Moreparticularly the present invention relates to 26,26,26,trifluoro-1α,25-dihydroxy-cholecalciferol, particularly in its25-epimeric forms, which are unexpectedly more potent than other vitaminD₃ derivatives and analogs in assays which predict vitamin D₃ -like,anti-proliferative and cellular differentiation inducing activities.Further aspects of the invention relate to the processes and novelintermediates utilized to prepare the desired end products andpharmaceutical preparations containing the biologically active endproducts.

DESCRIPTION OF THE INVENTION

The novel trifluoro compounds of the present invention are convenientlyprepared by synthesis from the novel indene sulfone intermediate of thestructure: ##STR1## where X is aryl, preferably phenyl and R₁, R₂, R₃each independently is lower alkyl, aryl or aralkyl, preferably R₃ is,(1,1-dimethylethyl) and R₁ and R₂ each are methyl.

In such synthesis the compounds of formula I above as an epimericmixture of arylsulfonyls at the 4 position are dearylsulfonylated withan alkali metal or with an alkali metal amalgam and in the presence ofan alkali metal phosphate. This reaction is conveniently carried out ina suitable inert organic solvent or solvent mixture such as a loweralkanol or cyclic ether or preferably mixtures thereof. A preferredreactant is sodium amalgam and dipotassium hydrogen phosphate, whilepreferred solvents include methanol, tetrahydrofuran and a mixture ofmethanol and tetrahydrofuran most preferably a 1:1 (v/v) mixture.

The alkali metal amalgam addition is carried out with cooling,preferably at temperatures below 0° C., most preferably at about -20° C.

Purification of the reaction products is carried out usingchromatographic procedures known per se. Thus, in preferred embodimentssilica column chromatography, provides the reaction product of formulaII below in purified form: ##STR2## where R₁, R₂ and R₃ are as above

In the next step of the synthesis, the protected compounds of formula IIare reacted with an epoxidation agent such as a peracid, most preferablytrifluoro-peracetic acid at reduced temperatures such as at 0° C.Preferably the reaction is carried out in the presence of an inorganicbase, such as for example a phosphate base most preferably dipotassiumhydrogen phosphate. Suitable inert organic solvents may be employed forthe reaction. Preferred for this purpose are halogenated alkyl or arylsolvents, most preferably chlorinated alkylenes such as methylenechloride. Under the conditions of the reaction part of the resultingproduct may consist of the desilylated product. Thus the product of thereaction is a mixture of the structures indicated below: ##STR3## whereR₄ is ##STR4## where R₁, R₂ and R₃ are as above.

Both the protected and unprotected compounds of formula III can be usedin the succeeding step. Thus, after purification from the above reactionmixture using methods known per se, such as flash chromatography eitheror both of a mixture of the two components of formula III can be treatedwith a chemical reducing agent, such as an alkali metal or metal-hydridepreferably lithium aluminum hydride in an inert organic solvent, mostpreferably an alkyl ether such as ethyl ether. The reaction ispreferably carried out under ambient conditions of temperature andpressure, most preferably under an inert atmosphere such as an argonatmosphere. There is thus produced the corresponding protected and/orunprotected compounds of the formula: ##STR5## where R₄ is as above.

The compounds of formula IV can be conveniently purified usingchromatographic procedures known per se, such as silica gelchromatography. It should be noted that the compounds of formula IVrepresent a mixture of epimers at the 5-position in the side chain.

The compounds of formula IV are then treated with a cation exchangeresin to deprotect any silylated compound. The mixture of epimers can beseparated, after purification by silica chromatography, by using highperformance liquid chromatogarphy on a silica column. The selection of asilica column is not critical. A suitable silica column for this purposeis sold under the trademark Magnum 9 Partisil-10 by Whatman Inc. ofClifton, N.J. although any conventional HPLC silica column may beemployed. To produce end products which are mixtures of epimers, thehigh performance liquid chromatography step can be eliminated.

The compounds of formula IV where R₄ is hydrogen are then oxidized usingan oxidative agent. Suitable oxidative agents useful in oxidation of the4-ring hydroxyl group include chromate salts, particularly with basicorganic amines, such as, for example, pyridinium halochromates,preferably pyridinium chlorochromate. The reaction is carried out underambient conditions of temperature and pressure using an inert solvent.Suitable inert solvents include the halogenated alkanes, preferably achloroalkene such as methylene chloride. There is thus obtained ketonesof the formula ##STR6## wherein Z is R-, S- or an R-, S- mixture

The ketones of formula V are then converted to their side chain hydroxyprotected forms without isolation. Thus ketones of formula V are treatedwith a trialkylsilylization agent, preferably a trimethylsilylizationagent to introduce a trimethylsilyl protecting group on the side chainhydroxy. A most preferred reagent for this purpose istrimethylsilylimidazole. The reaction is conveniently carried out atambient temperature, preferably under an inert atmosphere.

The resulting protected compounds of the formula: ##STR7## wherein Y isalkyl and Z is R-, S- or an R-, S-mixture are reacted with the carbanionof[3S-(3α,5β,Z)]-2-[2-methylene-3,5-bis[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyldiphenylphosphine oxide followed by removal of the trialkylsilyl protectinggroups to yield the desired end products of the invention, particularly:(A) 26,26,26,-trifluoro-1α,25S-dihydroxy cholecalciferol (B)26,26,26-trifluoro-1α,25R-dihydroxy cholecalciferol.

The above reaction is carried out at reduced temperatures e.g. below-50° C., most preferably at about -78° C. using an inert atmosphere suchas for example an argon atmosphere. A suitable inert solvent may beemployed in carrying of this reaction, for example, a cyclic ether, mostpreferably tetrahydrofuran. The conversion of the phosphine oxide to thecorresponding carbanion is readily accomplished by treating thephosphine oxide with an alkyl lithium such as preferably n-butyllithium.

Removal of the trialkylsilyl protecting group can readily beaccomplished by treating the reaction product with a cation exchangeresin under ambient conditions in a suitable solvent such as ahalogenated alkane, or preferably methylene chloride.

The final products of the invention can be purified by procedures knownper se such as, for example, by use of silica gel chromatography.

The novel starting materials of formula I used in the above describedsynthesis are readily obtainable from known compounds available in theart. Thus, for example,[1R-[1β,[αS*,βS*],3aα,4aβ,7aβ]]-octahydro-β,7a-dimethyl-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-α-ethenyl-1H-indene-1-ethanolcan be converted into[1R-[1β(R*),3aα,4β,7aβ]]-1-(4-chloro-1-methyl-2-butenyl)-octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indeneby treatment with thionyl chloride in the presence of pyridine. Theresulting allylic chloride is then reacted with an aryl sulfinic acidsalt, preferably benzene sulfinic acid sodium salt to yield thecorresponding arylsulfonyl compound, e.g.,[1R-[1β(R*),3aα,4β,7aβ]-1-[4-phenylsulfonyl)-1-methyl-2-butenyl)octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene.The aforesaid indene is then catalytically hydrogenated using apalladium-on-carbon catalyst to provide the corresponding side chainsaturated compound [1R-[1β(R*),3aα,4β,7aβ]]-1-[4-(phenylsulfonyl)-1-methylbutyl)-octahydro-4-[[1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene.The completion of the side-chain to produce a compound of formula I isaccomplished by reacting the carbanion of the aforesaid sulfonylcompound, formed by treatment with n-butyl lithium or lithiumdiisopropylamide, with trifluoroacetone.

The specific details of each of the reaction steps used in producing theintermediates of formula I according to the synthetic steps outlinedabove are set forth in the accompanying Examples below.

The compounds of the present invention can be administered in dosagesthat are in the range of about 0.10-3.0 micrograms/per day for thetreatment of such disease states as osteoporosis, osteodystrophy,steroid induced osteopenia, hypoparathyroidism, hypophosphatemic ricketsand hypophosphatemic osteomalacia which are characterized by lower thannormal levels of endogeneously produced 1α,25-dihydroxycholecalciferol.The compounds of the invention are also powerful specific inducers ofcell differentiation and inhibitors of cell proliferation. Thus, suchcompounds are useful agents in the treatment of proliferative diseasestates such as leukemia. Preferable dosage ranges are 0.25-2.0micrograms per day for the treatment of the aforementioned diseasestates. The compounds of the invention can be administered orally,subcutaneously, intramuscularly, intravenously, intraperitoneally ortopically.

The aforesaid products of the invention can be formulated intocompositions such as tablets, capsules, and the like, or elixers fororal administration, or in sterile solutions or suspensions forparenteral administration for the treatment of the aforementioneddisease states. About 0.10-3.0 micrograms, preferably 0.25-2.0micrograms, is compounded with a pharmaceutically acceptable vehicle,carrier, excipient, binder, preservative, stablizer, flavor, and thelike, in a unit dosage as called for by accepted pharmaceuticalpractice. The amount of active substance in the foregoing compositionsor preparations is in the range previously indicated.

Illustrative of the adjuvants which may be incorporated into capsules,and the like are the following: a binder such as gum tragacanth, acacia,corn starch, or gelatin; an excipient such as calcium phosphate; adisintegrating agent such as corn starch, potato starch, algenic acid,and the like; a lubricant such as magnesium stearate, a sweetening agentsuch as sucrose, lactose, or saccharin; a flavoring agent such aspeppermint, oil of wintergreen, or cherry. Various other materials maybe present as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets may be coated with shellac, sugar, orboth. A syrup or elixir may contain the active compound, sucrose as asweetening agent, methyl and propyl parabens as preservatives, a dye,and a flavoring such as cherry or orange flavor.

The above products of the invention can be administered for thetreatment of milk fever in pregnant ruminant animals prior toparturation in dosages in the range of 25-200 micrograms/day usingconventional formulations.

Sterile compositions for injection and/or topical administration can beformulated according to conventional practice by dissolving orsuspending the respective products of the invention in a vehicle such asa 10-20% ethanol-water mixture, a 10-20% propylene glycol-water mixturea naturally-occurring vegetable oil, such as sesame oil, peanut oil,cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyloleate or the like. For example, a suitable formulation for intravenousinjection would be 2-3 ml of a 10-20% ethanol-water solution or a 10-20%propylene glycol-water mixture containing 25-200 micrograms of theproducts of the invention. Such a formulation would preferably contain200-1000 micrograms of the products of the invention. Exemplary of asuitable formulation for topical administration would be a vegetable oilsolution or suspension containing 25-200 micrograms of the products ofthe invention. Such a formulation would preferably contain 200-1000micrograms of a product of the invention.

The aforesaid products of the invention can also be formulated for oraladministration by incorporation of 25-200 micrograms of such productinto fatty acid pellets.

The subject products may also be formulated for intramuscular injectionby suspension of 100-1500 micrograms of such product in a vehicle suchas a vegetable oil, an ethanol-water solution containing from 80-95%ethanol or a propylene glycol-water solution containing from 80-95%propylene glycol.

Buffers, preservatives, antioxidants and the like can be incorporatedinto the foregoing formulations as required.

As used throughout the specification and the appended claims, the term"lower alkyl" refers to a monovalent substituent consisting solely ofcarbon and hydrogen of from 1 to 8 carbon atoms which may be straight orbranched-chain. Examples of lower alkyl groups are methyl, ethyl,n-propyl, i-propyl, tert.-butyl, hexyl, heptyl, octyl and so forth. Theterm "lower alkylene group" refers to a divalent substituent consistingsolely of carbon and hydrogen of from 1 to 8 carbon atoms which may bestraight or branched-chain and whose free valences are attached to twodistinct groups. Examples of alkylene groups are methylene, ethylene,propylene and so forth. The term "lower alkoxy" refers to a lower alkylgroup attached to the remainder of the molecule by oxygen. The term"aralkyl" refers to aryl lower alkyl groups such as benzyl, phenethyland the like.

Examples of alkoxy groups are methoxy, ethoxy, isopropoxy, tert.-butoxyand so forth. The term "phenyl alkoxy" refers to an alkoxy group whichis substituted by a phenyl ring. Examples of phenyl alkoxy groups arebenzyloxy, 2-phenylethoxy, 4-phenylbutoxy and so forth. The term"alkanoyloxy group" refers to the residue of an alkylcarboxylic acidformed by removal of the hydrogen from the hydroxyl portion of thecarboxyl group. Examples of alkanoyloxy groups are formyloxy, acetoxy,butyryloxy, hexanoyloxy and so forth. The term "aryl" means phenyl andsubstituted phenyl. The term "substituted" as applied to "phenyl" refersto phenyl which is substituted with one or more of the following groups:alkyl, halogen (i.e., fluorine, chlorine, bromine or iodine), nitro,cyano, trifluoromethyl and so forth. The term "alkanol" refers to acompound derived by protonation of the oxygen atom of an alkoxy group.Examples of alkanols are methanol, ethanol, 2-propanol,2-methyl-2-propanol and the like. The term "alkali metal" refers tolithium, sodium and potassium.

In the formulas presented herein, the various substituents areillustrated as jointed to the steroid nucleus by one of these notations:a solid line (--) indicating a substituent which is in the β-orientation(i.e., above the plane of the molecule), a dotted line (---) indicatinga substituent which is in the α-orientation (i.e., below the plane ofthe molecule), or a wavy line indicating a substituent which may be inthe α- or β-orientation. The formulae have all been drawn to show thecompounds in their absolute stereochemical configurations. Since thestarting materials are derived from a naturally occurring steroid, theproducts exist in the single absolute configuration depicted herein.However, the processes of the present invention are intended to apply aswell to the synthesis of steroids of the "unnatural" and racemic series,i.e., the enantiomers of the compounds depicted herein and mixtures ofboth. Thus, one may begin the synthesis utilizing "unnatural" or racemicstarting materials to prepare "unnatural" or racemic products,respectively.

The nomenclature adopted to define absolute configuration ofsubstituents bound to carbon atom 24 of the steroid nucleus is describedin the Journal of Organic Chemistry, 34, 2849 (1970) under the title"IUPAC Tentative Rules for the Nomenclature of Organic Chemistry.Section E. Fundamental Stereochemistry.

EXAMPLE 1 Preparation of[1R-[1β(R*),3aα,4β,7aβ]]-1-(4-chloro-1-methyl-2-butenyl)-octahvdro-4-[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene

A solution of 2.9 g (8.22 mmol) of[1R-[1β,[αS*,βS*],3aα,4aβ,7aβ]]-octahydro-β,7a-dimethyl-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-α-ethenyl-1H-indene-1-ethanolin 100 mL of anhydrous ether was cooled at 0° C. and treated dropwiseand under argon with 2.76 mL (37.84 mmol) of thionyl chloride, followedby 0.276 mL of pyridine. The mixture was allowed to stir at 0° C. for 2h, then it was quenched by addition of 50 mL nf a 2N sodium potassiumtartrate solution. The ether phase was separated and the aqueous phaseextracted with ethyl acetate. The combined organic phases were washedwith 1N hydrochloric acid, water, 2N potassium bicarbonate solution andbrine, dried (Na₂ SO₄) and evaporated. The solvent evaporated in vacuoand the residue purified by rapid chromatography on silica (eluent:hexane-ethyl acetate, 19:1(v:v)) to give 2.9 g (95% yield) of the titlecompound, as a low melting solid.

EXAMPLE 2 Preparation of[1R-[1β(R*),3aα,4β,7aβ]]-1-[4-(phenylsulfonyl)-1-methyl-2-butenyl)-octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene

A solution of 2.9 g (7.81 mmol) of[1R-[1β(R*),3aα,4β,7aβ]]-1-(4-chloro-1-methyl-2-butenyl)-octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indenein 130 mL of hexamethylphosphboramide was treated with 10.1 g (61.52mmol) of benzene sulfinic acid sodium salt and stirred at roomtemperature under argon for 24 h. Ice water was then added (130 mL) and,after stirring for 30 min, the mixture was extracted with ethyl acetate.The combined extracts were washed with water (6x) dried (Na,SO₄),evaporated to dryness and the residue purified by rapid chromatographythrough silica, eluting with hexane-ethyl acetate (39:1 (v:v)) to give3.5 g (94% yield) of the above-captioned product as a low melting solid.

EXAMPLE 3 Preparation of[1R-[1β(R*),3aα,4β,7aβ]]-1-[4-(phenylsulfonyl)-1-methylbutyl)-octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene

A solution of 1.00 g (2.10 mmol) of[1R-[1β(R*),3aα,4β,7aβ]]-1-[4-(phenylsulfonyl)-1-methyl-2-butenyl)octahydro-4-[[(1,1-dimethylethyl)-dimethylsilyl]-oxy]-7a-methyl-1H-indenein 40 mL of ethanol was hydrogenated at room temperature and undernormal pressure over 350 mg of 10% palladium-on-carbon. After 2 h, thecatalyst was filtered and the filtrate evaporated to dryness to give1.00 g of the above captioned product.

EXAMPLE 4 Preparation of[1R-[1β(1R*),3aα,4β,7aβ]]-Octahydro-1-6,6,6-trifluoro-5-hydroxy-1,5-dimethyl-4-(phenylsulfonyl)hexy]-4-[[(1,1-dimethylethyl)dimethylsilyl]-oxy]-7a-methyl-1H-indene

A solution of 0.440 mL (3.14 mmol) of diisopropylamine in 10 mL ofanhydrous tetrahydrofuran was cooled at 0° C. and treated dropwise underargon with 1.87 mL (2.99 mmol) of a 1.6 molar solution of n-butyllithiumin hexane. After stirring for 15 min at 0° C., the resulting solutionwas cooled at -78° C. and diluted with 10 mL of anhydroustetrahydrofuran. It was then treated dropwise with a solution of 1.00 g(2.09 mmol) of[1R-[1β(R*),3aα,4β,7aβ]]-1-[4-(phenylsulfonyl)-1-methylbutyl)-octahydro-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-inolenein 15 mL of tetrahydrofuran and stirred at -78° C. for 5 min, then at 0°C. for 30 min. After cooling again at -78° C., the mixture was treatedwith 0.50 mL (5.59 mmol) of 1,1,1-trifluoroacetone and stirred at thesame temperature for 1.5 h. It was then quenched by addition of 30 mL ofa 1:1 mixture of 2N sodium potassium tartrate and 2N potassiumbicarbonate solution, allowed to come at room temperature and extractedwith methylene chloride. The combined organic extracts were washed withbrine, dried (Na₂ SO₄) and evaporated to dryness. The residue waspurified by flash chromatography through silica (eluent hexane-ethylacetate, 39:1 (v:v)) to give 0.71 g of the above captioned product as acolorless oil.

EXAMPLE 5 Preparation of[1R-[1β(R*),3aα,4β,7aβ]]-Octahydro-1-[6,6,6-trifluoro-1,5-dimethyl-4-hexenyl]-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene

A solution of 0.56 g (0.95 mmol) of the sulfone product of Example 4(epimeric mixture) in 18 mL of tetrahydrofuran and 18 mL of methanol wastreated with 10 g of dipotassium hydrogen phosphate and after cooling at-20° C., with 11 g of 6% sodium amalgam. After stirring the resultingmixture at -20° C. for 10 min, 30 mL of brine was added, allowed to cometo room temperature and extracted with ethyl acetate. The combinedorganic extracts were washed with brine, dried (Na₂,SO₄,) and evaporatedto dryness. The residue was purified by chromatography through silica(eluting with hexane-ethyl acetate, 39:1 (v:v)) to give 0.40 g of theabove-captioned olefin as a colorless oil (mixture of geometricalisomers).

EXAMPLE 6 Preparation of[1R-[1β(1R*),3aα,4β,7aβ]]-Octahydro-1-[6,6,6-trifluoro-1,5-dimethyl-24,25-epoxyhexyl]-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene.

A solution of 0.260 mL (1.84 mmol) of trifluoroperacetic anhydride in 5mL of anhydrous methylene chloride was cooled at 0° C., treated with0.052 mL (1.89 mmol) of 90% hydrogen peroxide and stirred for 30 min.The resulting solution was rapidly added to 400 mg of the product ofExample 5 (0.92 mmol) and 1.5 g of dipotassium hydrogen phosphate in 5mL of methylene chloride at 0° C. After addition, the mixture wasallowed to come to room temperature for 30 min, quenched with 5 mL of a10% aqueous solution of sodium sulfite, diluted with 10% aqueous sodiumbicarbonate solution and extracted with methylene chloride. The combinedorganic extracts were washed with brine, dried and evaporated to give190 mg of pure protected product (mixture of epimers) and 140 mg ofdesilylated product. After flash chromatography purification, bothproducts can be used in the next step.

EXAMPLE 7 Preparation of[1R-[1β(1R*)-3aα,4β,7aβ]]Octahydro-1-[6,6,6-trifluoro-5-hydroxy-1,5-dimethylhexyl]-4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-7a-methyl-1H-indene

To a suspension of 100 mg (2.63 mmol) of lithium aluminum hydride in 3mL of anhydrous ether, a solution of 190 mg (0.42 mmol) of the protectedproduct of Example 6 in 2 mL of ether was rapidly added at roomtemperature and under argon atmosphere and the resulting mixture stirredfor 24 h. It was then quenched by careful addition of 2N aqueous sodiumpotassium tartrate, extracted with ether and the combined organicextracts washed with brine, dried and evaporated to give, afterpurification over silica (eluent, hexane-ethyl acetate, 19:1 (v:v)), 175mg of pure captioned product (mixture of epimers) as a thick oil.

EXAMPLE 8 Preparation of[1R-[1β(1R*,5R*)-3aα,4β,7aβ]]-Octahydro-1-[6,6,6-trifluoro-5-hydroxy-1,5-dimethylhexyl]-7a-methyl-1H-inden-4-oland [1R-[1β(1R*,5S*)-3aα,4β,7aβ]]Octahydro-1-[6,6,6-trifluoro-5-hydroxy-1,5-dimethylhexyl]-7a-methyl-1H-inden-4-ol

A solution of 175 mg (0.39 mmol) of the product of Example 7 (mixture ofepimers) in 9 mL of methanol was treated with 2.8 g of AG 50W-X4 cationexchange resin (200-400. mesh, Bio-Rad Laboratory, Richmond, CA) andstirred at room temperature for 12 days. The resin was removed byfiltration, washed with methanol and the combined filtrates evaporatedto dryness. The residue was purified by rapid filtration through silica(eluent: hexane-ethyl acetate, 9:1 (v:v)), then the two epimers wereseparated by high performance liquid chromatography, using a Magnum 9Partisil-10 silica column (Whatman Inc., Clifton, N.J.) and eluting withchloroform/ethyl acetate (5:1 (v:v)) to, give 65 mg of crystalline 5R*product, m.p. 110°-111° C., [α]²⁵ D+40.3° (c 0.2 in chloroform)retention time 16.4 minutes, the structure of which was confirmed byX-ray crystallographic analysis and 55 mg of 55* product, [α]²⁵ D+31.6°(c 0.2 in chloroform), as a thick oil, retention time 15.2 minutes.

EXAMPLE 9 Preparation of [1R-[1β(1R*,5R*),3aα,4β,7aβ]]-Octahydro-1-6,6,6-trifluoro-5-(trimethylsilyloxy)-1,5-dimethylhexyl]-7a-methyl-1H-inden-4-one

A solution of 50 mg (0.15 mmol) of the 5R* diol product of Example 8 in2 mL of methylene chloride was added to a slurry of 97 mg (0.45 mmol) ofpyridinium chlorochromate in 4 mL of methylene chloride and theresulting mixture stirred at room temperature for 1.5 h. It was thendiluted with 10 mL of ether, stirred for 15 min, filtered throughCelite® and the residue of the filtration triturated several times withether and the trituration extracts combined, filtered and evaporated todryness. The residue was purified by flash chromatography (eluent:hexane-ethyl acetate, 3:1 (v:v)) to give 49 mg of product. This wasdissolved in 4 mL of anhydrous methylene chloride, treated with 0.13 mL(0.89 mmol) of trimethylsilylimidazole and stirred at room temperatureunder argon for 22 h. After addition of 0.5 mL of water, the mixture wasstirred for an additional 20 min, then diluted with water and extractedwith ethyl acetate. The organic extracts were washed with water andbrine, dried (Na₂, SO₄) and evaporated to dryness. The residue waspurified by flash chromatography (eluting with hexane-ethyl acetate, 4:1(v:v)) to give 51 mg of captioned product as a thick oil.

EXAMPLE 10 Preparation of[1R-[1β(1R*,5S*),3aα,4β,7aβ]]-Octahydro-1-[6,6,6-trifluoro-5-(trimethylsilyloxy)-1,5-dimethylhexyl]-7a-methyl-1H-inden-4-one

Following the procedure described in Example 9, 56 mg of 55* diolproduct of Example 8 was converted to 59 mg of the above-captionedproduct.

EXAMPLE 11 Preparation of26,26,26-Trifluoro-1α,25R-dihydroxycholecalciferol

A solution of 141 mg (0.23 mmol) of[3S-(3α,5β,Z)]-2-[2-methylene-3,5-bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyldiphenylphosphine oxide in 6 mL of anhydrous tetrahydrofuran was cooled at -78°C. and treated dropwise and under argon with 0.137 mL (0.22 mmol) of a1.6 molar solution of n-butyllithium in hexane. After stirring for 5min, a solution of 51 mg (0.13 mmol) of the ketone product of Example 9in 1.5 mL of tetrahydrofuran was added dropwise to the deep orangephosphinoxy carbanion solution and the resulting mixture stirred at -78°C. for 1.5 h. It was then treated with 3 mL of a 1:1 (v:v) mixture of 2Npotassium sodium tartrate and 2N potassium bicarbonate solution, allowedto come to room temperature, diluted with water and extracted with waterand extracted with ethyl acetate. The combined organic layers werewashed with brine, dried and evaporated to dryness. The residue waspurified by fast filtration through silica eluted with hexane-ethylacetate, 20:1 (v:v), then dissolved in 0.5 mL of methylene chloride and5 mL of methanol and stirred at room temperature over-night with 2 g ofAG 50W-X4 cation exchange resin (Bio-Rad Laboratories, Richmond,Calif.). After filtration and evaporation of the solvent, the residuewas purified by flash chromatography, using hexane-ethyl acetate (1:2)as eluent to give 23 mg of product as a white amorphous powder, ¹ H NMR(400 MHz, CD₃ OD) 0.60 (s, 3H), 0.99 (d, J=6.4, 3H), 1.30 (s, 3H), 4.15(m, 1H), 4.38 (m, 1H), d 4.90 (br s, 1H), 5.31 (br s, 1H), 6.10 (dm,J=11.2 Hz, 1H), 6.34 (d, J=11.2 Hz, 1H).

EXAMPLE 12 Preparation of26,26,26-trifluoro-1α,25S-dihydroxycholecalciferol

Following the procedure of Example 11, 58 mg of the ketone product ofExample 10 were converted to 59 mg of26,26,26-trifluoro-1α,25S-dihydroxycholecalciferol.

¹ H NMR (400 MHz, CD₃ OD) 0.58 (s, 3H), 0.97 (d, J=6.8 Hz, 3H), 1.28 (s,3H), d 4.13 (m, 1H), d 4.36 (m, 1H), 4.90 (br s, 1H), 5.29 (br s, 1H),6.09 (d, J=11.2 Hz, 1H), 6.32 (d, J=11.2 Hz, 1H).

EXAMPLE 13

    ______________________________________                                        Item Ingredients   mg/capsule                                                 ______________________________________                                        1.   product of the                                                                              0.00010   0.00025 0.00050                                       invention                                                                2.   Polyethylene glycol                                                                         200.00    200.00  200.00                                        400 (PEG 400)                                                            3.   butylated hydroxy                                                                           0.100     0.100   0.100                                         anisole (BHA)                                                            4.   ascorbyl palmitate                                                                          1.00      1.00    1.00                                     ______________________________________                                        Procedure:                                                                    Dissolve items 1, 3 and 4 in item 2, under a blanket of nitrogen              and encapsulate.                                                              ______________________________________                                    

EXAMPLE 14

    ______________________________________                                        Item Ingredients   mg/capsule                                                 ______________________________________                                        1.   product of the                                                                              0.00010   0.00025 0.00050                                       invention                                                                2.   Polyethylene glycol                                                                         200.00    200.00  200.00                                        400 (PEG 400)                                                            3.   butylated hydroxy                                                                           0.100     0.100   0.100                                         anisole (BHA)                                                            4.   ascorbyl palmitate                                                                          1.00      1.00    1.00                                     ______________________________________                                        Procedure:                                                                    Dissolve items 1, 3 and 4 in item 2, under a blanket of nitrogen              and encapsulate.                                                              ______________________________________                                    

    ______________________________________                                        Item    Ingredients                                                           ______________________________________                                        1.      product of the invention                                                                       0.10   mg  0.50  mg                                  2.      95% ethanol-5% water                                                                           2.00   ml  3.00  ml                                  ______________________________________                                        Procedure                                                                     Dissolve item 1 in item 2 under a blanket of nitrogen                         and inject intramuscularly.                                                   ______________________________________                                    

EXAMPLE 15

Subject: Anti-proliferative and differentiation-inducing effects of26,26,26,-trifluoro-1α,25R-dihydroxycholecalciferol and26,26,26,-trifluoro-1α,25S-dihydroxycholecalciferol.

General experimental description

Cultures of HL-60 cells were established in the absence (control) orpresence of various concentrations of the test compounds. After a 4-dayincubation period, the cultures were evaluated for proliferation oftumor cells, tumor cell viability, and cellular differentiation.Proliferation was assessed by directly enumerating the increased numberof tumor cells resulting from incubation. Viability was determined bydye exclusion technique to learn whether any of the compounds werelethal to cultured HL-60 cells. Cellular differentiation was evaluatedby determining the number of cells which had acquired the enzymesnecessary to support a respiratory burst such activity beingcharacteristic of mature macrophages and granulocytes.

Methods

Tissue culture medium used in these experiments was RPMI-1640supplemented prior to use to 10% v/v with fetal bovine serum (heatinactivated at 56° C. for 30 minutes), to 130 units per ml withpenicillin and 130 μg per ml with streptomycin, and to an additional 1.6millimolar with L-glutamine.

Experimental compounds were dissolved in sufficient ethanol to yieldstock solutions of 1×10⁻³ molar. Reduced lighting was employed whenworking with compounds and stock solutions were stored in the dark at-20° C. in an argon atmosphere. Compounds were diluted with tissueculture medium and added to flasks containing HL-60 cells to achieve thefinal concentrations described in each experiment.

The promyelocytic (HL-60) tumor cell line was derived from a patientwith acute promyelocytic leukemia. HL-60 cells were maintained in liquidculture by serial weekly passage in tissue culture medium. In anyexperiment, three replicate flasks were incubated without compound(control) or in the presence of varying concentrations of compound.After 4 days of incubation at 37° C. in a humidified atmosphere of 5%CO₂ in air, cultures were evaluated for tumor cell proliferation,viability and differentiation.

Quantitation of proliferation was done by enumerating the number ofHL-60 cells in each individual flask (3 flasks per experimental point)using a model ZBI Coulter Counter. Results are shown as the number ofcells per ml of tissue culture medium expressed as the mean±standarddeviation and as percent reduction of cell number calculated accordingto the formula: ##EQU1## Experimental cultures with the same or slightlygreater cell numbers than control cultures are reported as zero percentreduction.

Viability of tumor cells was determined by the method of trypan blue dyeexclusion. Cells in tissue culture medium were added to a four-foldlarger volume of 0.4% trypan blue in saline. Cells were scored as viableupon microscopic examination if they excluded dye and as dead if theywere stained blue. The viability of cells from all experimental cultureswas never less than that from control cultures indicating that thecompounds tested were not toxic to HL-60 cells in the concentrationsemployed.

Quantitation of differentiated cells was done by the biochemical methodof nitroblue tetrazolium (NBT) reduction. Sufficient cells were pooledfrom replicate cultures, centrifuged at 220×g, washed once with serumfree tissue culture medium, and resuspended to 1×10⁶ cells per ml inCa⁺⁺ --Mg⁺⁺ -deficient phosphate buffered saline (prepared bysupplementing Ca⁺⁺ --Mg⁺⁺ -free phosphate buffered saline (PBS) to 10%v/v with heat-inactivated fetal bovine serum). Nitroblue tetrazolium wasdissolved at 1 mg per ml in Ca⁺⁺ --Mg⁺⁺ -deficient PBS with gentleheating and mixing. Tetradecanoyl phorbol acetate (TPA) was dissolved at1 mg per ml in ethanol and stored at -20° C. Just prior to use, aworking solution of TPA was prepared by diluting the stock concentration100-fold with Ca⁺⁺ --Mg⁺⁺ -deficient PBS. The test was done in 12×75 mmtubes by adding 0.5 ml Ca⁺⁺ --Mg⁺⁺ -deficient PBS, 1.0 ml of HL-60cells, 0.5 ml of NBT solution, and 0.02 ml of the working TPA solution.After mixing, the tubes were incubated in a 37° C. water bath for 25minutes then transferred to ice. Undifferentiated and differentiatedcells present in any sample were determined microscopically by surveying200-400 cells per sample. Cells without pigmented granules (clear cells)were judged to be undifferentiated while those containing greater than 3blue-black formazan granules were scored as differentiated. Generally,differentiated cells were intensely pigmented clearly indicating theenzymatic conversion of NBT to formazan. Results are expressed as thepercentage of differentiated cells present in any sample as calculatedaccording to the formula: ##EQU2##

The results obtained are summarized in Table I.

                                      TABLE I                                     __________________________________________________________________________    ANTI-PROLIFERATIVE AND DIFFERENTIATION-INDUCING EFFECTS OF                    26,26,26-TRIFLUORO-1α,25R-DIHYDROXYCHOLECALCIFEROL AND                  26,26,26-TRIFLUORO-1α,25S-DIHYDROXYCHOLECALCIFEROL ON                   HL-60 Cells, IN VITRO                                                                        Proliferation.sup.c                                                                         Differentiation                                  Concentration.sup.a,b                                                                        HL-60 Cells                                                                          % Reduction                                                                          NBT Reduction                                    and Compound   per ml of cell                                                                              formazan "+" cells                               (× 10.sup.-9 molar)                                                                    × 10.sup.-4                                                                    number total cells counted                                                                     % "+"                                  __________________________________________________________________________    None (medium control)                                                                        84.7 ± 4.3                                                                        --      3/368    <1                                     Vehicle (0.1% ethanol)                                                                       78.9 ± 4.9                                                                        0       4/356    1                                      26-(F).sub.3 -1α,25R-(OH).sub.2 D.sub.3 1                                              59.1 ± 1.9                                                                        30      81/353   23                                     26-(F).sub.3 -1α,25R-(OH).sub.2 D.sub.3 10                                             27.2 ± 0.3                                                                        68     299/318   94                                     26-(F).sub.3 -1α,25R-(OH).sub.2 D.sub.3 100                                            22.9 ± 1.0                                                                        73     360/367   98                                     26-(F).sub.3 -1α,25R-(OH).sub.2 D.sub.3 1000                                           22.0 ± 0.7                                                                        74     340/344   99                                     26-(F).sub.3 -1α,25S-(OH).sub.2 D.sub.3 1                                              60.2 ± 3.9                                                                        29      84/334   25                                     26-(F).sub.3 -1α,25S-(OH).sub.2 D.sub.3 10                                             27.0 ± 1.4                                                                        68     338/354   96                                     26-(F).sub.3 -1α,25S-(OH).sub.2 D.sub.3 100                                            22.3 ± 2.6                                                                        74     323/330   98                                     26-(F).sub.3 -1α,25S-(OH).sub.2 D.sub.3 1000                                           20.2 ± 0.5                                                                        76     353/358   99                                     __________________________________________________________________________     .sup.a Vehicle concentration in all experimental cultures was 0.1% v/v,       ethanol.                                                                      .sup.b 26(F).sub.31α,25R(OH).sub.2D.sub.3 is                            26,26,26trifluoro-1,25R-dihydroxycholecalciferol and                          26(F).sub.31α,25S(OH).sub.2D.sub.3 is                                   26,26,26trifluoro-1α,25Sdihydroxy-cholecalciferol.                      .sup.c The cell viability in all cultures was greater than 97%. All           cultures were initiated with 2 × 10.sup.4 HL60 cells per ml. Shown      for each point is the mean ± S.D.                                     

RESULTS

The experiment described in Table 1 documents that the product of theinvention inhibited the proliferation of human promyelocytic tumor cells(HL-60), in vitro, in a dose-dependent fashion. The lowest concentrationtested (1×10⁻⁹ molar) was markedly effective and increasingconcentrations improved the anti-proliferative effect. With theexperimental methods employed, the maximal anti-proliferative effect wasseen at concentrations in excess of 10×10⁻⁹ molar since higherconcentrations of 100 and 1000×10⁻⁹ molar were only slightly moreeffective. Cells from each of the cultures were assessed fordifferentiation by the method of NBT reduction as also shown in Table I.In contrast to cells from control cultures, a portion of the cells fromexperimental cultures had synthesized the enzymes necessary to reduceNBT to formazan and were thus judged to be differentiated. Moreover, theproportion of differentiated cells present in any culture was directlyrelated to the concentration of compound present during incubation.Similar to the anti-proliferative effect, the differentiation-inducingeffect of the compounds was maximal at approximately 10×10⁻⁹ molar.

Taken together, these data indicate that the products of the inventionrestrained the proliferation of human promyelocytic tumor cells eventhough they were not toxic to the cells. Furthermore, cells cultured inlow concentrations of the compounds (1×10⁻⁹ to 10×10⁻⁹ molar) wereinduced to differentiate toward a more mature cell type as evidenced bythe acquisition of enzyme activity. It is expected, then, that each ofthese compounds is useful as a unique approach to the management ofclinical diseases which owe in part to aberrant cellular proliferationand/or differentiation. Exemplary to this issue is the process ofneoplastic disease which owes to a perturbation of the normal processesof cellular differentiation.

We claim:
 1. 26,26,26-trifluoro-1α,25S-dihydroxycholecalciferol.
 2. Apharmaceutical composition suitable for oral administration saidcomposition comprising a therapeutically effective amount of26,26,26-trifluoro-1α, 25S-dihydroxycholecalciferol and a conventionalpharmaceutical carrier material suitable for oral administration.
 3. Apharmaceutical composition suitable for parenteral administration saidcomposition comprising a therapeutically effective amount of26,26,26-trifluoro-1α, 25S-dihydroxycholecalciferol and a conventionalpharmaceutical carrier material suitable for parenteral administration.